A method for assaying endotoxin (hereinafter referred to as Et) using an extract of horseshoe crab blood cells (amebocytes), i.e., a limulus amebocyte lysate (hereinafter simply referred to as a lysate) is well known as a limulus test. The assay comprising the reaction of the lysate is called a limulus reaction. A limulus test has so high sensitivity that is widely employed in pyrogen check of drugs and water, diagnostic use, and the like. The limulus test is based on coagulation of a lysate in the presence of a trace amount of Et. The latest biochemical study has elucidated the fact that the limulus reaction is composed of stepwise activation of several coagulation factors (see Takanori Nakamura, et al., Japanese Journal of Bacteriology, 38, 781-803 (1983)).
The limulus reaction is illustrated below with respect to a lysate from Tachypleus tridentatus by referring to FIG. 1. On Et addition to a lysate, an Et-sensitive factor (factor C; molecular weight: 123,000) in the lysate is activated. The activated factor C limitedly hydrolyzes factor B (molecular weight: 64,000) at a specific site to produce activated factor B. The activated factor B activates proclotting enzyme (molecular weight: 54,000) to convert into clotting enzyme. The clotting enzyme limitedly hydrolyzes coagulogen (coagulant protein; molecular weight: 19,723) at the specific sites in the loop crosslinked by a disulfide linkage, i.e., intermediate between . . . Arg.sup.18 and Thru.sup.19 . . . and intermediate between . . . Arg.sup.46 and Gly.sup.47 . . . to release peptide C (28 amino acid residues) represented by H-Thru.sup.19 . . . Arg.sup.46 -OH while converting the remaining part into coagulin gel. Thus, the limulus reaction is composed of a series of reactions (cascade reaction caused by Et is hereinafter referred to as factor C system reaction).
The above-mentioned cascade reaction of a lysate is induced by not only Et but also a (1.fwdarw.3)-.beta.-D-glucan (hereinafter simply referred to as a .beta.-glucan). That is, factor G in FIG. 1 is activated by a .beta.-glucan, the activated factor G converts proclotting enzyme into clotting enzyme, and clotting enzyme acts on coagulogen to produce coagulin gel in the same manner of Et as described above (cascade reaction caused by a .beta.-glucan is hereinafter referred to as factor G system reaction).
The clotting enzyme produced through the cascade reaction is also capable of hydrolyzing an amide linkage of a synthetic peptide substrate separately added to the reaction system, such as t-butoxycarbonyl-leucyl-glycyl-arginine-paranitroanilide (Boc-Leu-Gly-Arg-pNA) to release paranitroaniline. Accordingly, Et or the .beta.-glucan can be quantitatively determined by measuring the absorbance of the thus released paranitroaniline.
It has been proposed to use a lysate from which factor G has been removed so as to specifically assay Et based on the factor C system only as disclosed in Obayashi T., et al., Clin. Chim. Acta, 149, 55-65 (1985).
However, this method involves extremely complicated operations for preparing a factor G-free system, including fractionation of a lysate by affinity chromatography using an affinity carrier having immobilized thereon dextran sulfate to remove a .beta.-glucan-sensitive factor, i.e., factor G, and reconstitution of factor C, factor B and proclotting enzyme. Et-specific assay is made by using the resulting factor G-free system and a synthetic peptide substrate for clotting enzyme. It is also known to use a combination of an Et-sensitive factor, i.e., factor C, and a synthetic peptide substrate for activated factor C (e.g., t-butoxycarbonyl-valyl-prolyl-arginine-paranitroanilide, hereinafter represented by Boc-Val-Pro-Arg-pNA) for Et-specific assay as disclosed in Nakamura T., et al., Eur. J. Biochem., 154, 511-521 (1986). This technique also requires complicated operations for separation and purification.
The above-mentioned conventional techniques of limulus assay are based on a liquid phase reaction system using a part or the whole of a lysate.
Samples to be assayed frequently contain various substances interfering with a limulus test and need to be previously treated for inactivation or removal of the interfering substances. For example, heavy metals or salts if present in high concentrations strongly interfere with the limulus reaction, making it impossible to obtain an accurate Et level. In these cases, the sample must be diluted with distilled water until no interference takes place, but the allowable degree of dilution is limited by the detection limit of the limulus test. Further, cases are often met in which a turbid or colored sample, even after being diluted, cannot be assayed. Blood samples or milk samples require complicated pretreatments. Thus, Et assays in these samples involve many problems to be solved.
Besides the liquid phase reaction systems, a system using a lysate immobilized to the wells of a polystyrene microplate is known (J. Clin. Microbiol., 17, 1050-1053 (1983)). However, since not only the Et-sensitive factor but .beta.-glucan-sensitive factor are simultaneously immobilized, the system cannot be used for Et-specific assay. A test paper prepared by impregnating filter paper with a lysate, a synthetic peptide substrate, etc. followed by drying is also proposed (JP-A-62-134563, the term "JP-A" as used herein means an "unexamined published Japanese patent application"). However, since the lysate-impregnated filter paper is not subjected to washing, it contains all the components of the lysate and is therefore unsuitable for Et-specific assay.